Neurotransmission disorders

ABSTRACT

There is disclosed a method for diagnosing neurotransmission or developmental disorders in a mammal comprising the step of detecting in a bodily fluid of said mammal autoantibodies to an epitope of the muscle specific tyrosine kinase (MuSK). One such method comprises a) contacting said bodily fluid with said MuSK or an antigenic determinant thereof; and b) detecting any antibody-antigen complexes formed between said receptor tyrosine kinase or an antigenic fragment thereof and antibodies present in said bodily fluid, wherein the presence of said complexes is indicative of said mammal suffering from said neurotransmission or developmental disorders. Also disclosed are kits for use in the diagnosis of neurotransmission and subsequent developmental disorders.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/311,575, with a 371(c) date of Jun. 6, 2003, which is the U.S.National Stage of International Application No. PCT/GB01/02661, filed onJun. 15, 2001, published in English, which claims priority under 35U.S.C. § 119 or 365 to Great Britain, Application No. GB 0014878.3,filed Jun. 16, 2000. The entire teachings of the above application(s)are incorporated herein by reference.

SUMMARY OF THE INVENTION

The present invention is concerned with neurotransmission disorders and,in particular, with a method of diagnosing such disorders in mammals.Also provided by the present invention are kits for use in saiddiagnosis.

Myasthenia gravis (MG) is a chronic autoimmune disorder of neuromusculartransmission resulting in muscle weakness. The key feature of weaknessdue to MG is its variability. Patients generally experience a waning ofstrength throughout the day with a tendency to fatigue later in the dayor even towards the end of a particular task. A symptom of MG is oftenocular weakness, causing ptosis (drooping eyelids) and/or diplopia(double vision). Other symptoms include leg weakness, dysphagia andslurred or nasal speech. Symptoms of weakness tend to worsen withvarious stressors, such as, exertion, heat and infection.

In 1960 it was discovered that MG was caused by antibodies against theacetyl choline receptor (AChR) and that it is therefore autoimmune inorigin. Today MG is one of the most characterized of neurologicaldisorders which has consequently lead to treatments which vastly improvethe length and quality of life of myasthenics. Approximately 10 peoplein every million of a population contract this disease in one year.There is no racial predominance and 75% of MG patients less than 40years of age are female and 60% of those older than 40 years are male.

Approximately 80% of patients with MG possess within their plasmaautoantibodies that are immunoprecitipatable with radiolabeled AChR. Theremaining 20% of MG patients do not, however, exhibit such antibodies intheir plasma but do have similar symptoms and respond to the sametherapies such as plasma exchange and immunosuppression. Accordingly, ithas not been established whether these patients have the same or adistinct and separate MG condition (3,4). Autoantibodies are naturallyoccurring antibodies directed to an antigen which an individual's immuneresponse recognizes as foreign even though that antigen actuallyoriginated in the individual. They may be present in the circulatorysystem as circulating free antibodies or in the form of circulatingimmune complexes bound to their target depending on the nature of theantigen concerned.

Human plasma from patients who were anti-AChR autoantibodies negative(AAAN or previously known as sero-negative MG), were investigated foralternative autoantibodies and one candidate autoantibody was that onefor the MuSK protein.

The present inventors surprisingly found that many of the 20% of MGpatients which do not exhibit any autoantibodies to AChR, instead haveIgG antibodies directed against the extracellular N-terminal domains ofMuSK, a receptor tyrosine kinase located on the cell. surface ofneuromuscular junctions, indicating that they are afflicted with a formof MG which has a different etiology from MG characterised bycirculating autoantibodies to AChR.

The MuSK protein has been sequenced and the protein characterisedrecently by Valenzuela et al. (International patent application numberPCT/US96/20696, published as W097/21811). It is a receptor tyrosinekinase (RTK) located on the cell surface of muscle cells at theneuromuscular junction.

Ligands bind to RTKs at the binding site on the extracellular side ofthe receptor, which induces transmission of a signal cascade tointracellular target proteins. RTKs are classified according to theirfunction and members of these families share high homology in theiramino acid sequence as well as functionality.

At the neuromuscular junction (NMJ) where the motor nerve axon dendritesmeet the muscle cell basal membrane, important physiological signals areexchanged between these adjacent cells. An example of this is thechemical transmitter acetyl choline which passes through the synapticcleft from the nerve cell, and is then rapidly and specifically bound bythe AChR at the muscle cell wall. This in turn begins a cascade ofevents which ultimately leads to contraction of the muscle cells.

The post synaptic structure at the muscle cell wall is termed the motorendplate which is densely packed with protein and lipid, thereby givingan electron dense appearance when observed by electron microscopy. Themuscle AChRs are present here, and it is believed that signaling givesrise to concentrations of proteins there by two mechanisms; one isaltered distribution of pre-existing membrane proteins and the other isby induction of localized transcription of specific genes only bysubsynaptic nuclei underlying the NMJ.

Development of the neuromuscular junction is initiated throughactivation of MuSK. Agrin isoforms, released from the motorneuron,trigger MuSK and muscle acetylcholine receptor (AChR) phosphorylationresulting in clustering of AChRs and other proteins of the postsynapticapparatus (1). Agrin's ability to cause AChR clustering in culturedmyotubes has been shown to be inhibited by anti agrin antibodies. It iscurrently accepted that agrin does not bind directly to MuSK, but via ahypothetical agrin-binding component termed Myotubule AssociatedSpecificity Component (MASC) (1,11). No disease associated with eitherMuSK, MASC, or agrins has been reported and their roles in adult musclehave not yet been elucidated.

It has already been shown that anti AChR autoantibody negative MG iscaused by humoral IgG antibodies: it can be successfully treated byplasma exchange and other immune therapies (5); transient neonatal MGwas reported in the newborn infant of one of the patients with anti-MuSKantibodies (17); and injection of immunoglobulin or IgG preparationsinto mice caused defects in neuromuscular transmission (5).

The present inventors have therefore now shown that anti-MuSK antibodieshave functional effects on agrin-induced AChR clustering in vitro, anddirect interference with this agrin/MuSK/AChR pathway may be animportant disease mechanism in vivo. MuSK is a relatively new member ofthe receptor tyrosine kinase (RTK) family. With very few exceptions (forexample, see 18), autoantibodies to RTKs have not been implicated inhuman disorders but the combination of large extracellular domains andfunctional activities make them attractive potential antigens in otherautoimmune conditions. Other members of the RTK family are mutated ininherited diseases, and somatic mutations have been found in varioustumors (19). MuSK may prove to be involved in congenital as well asacquired muscle disorders.

Therefore, there is provided by a first aspect of the present inventiona method of diagnosing neurotransmission disorders in a mammalcomprising the step of detecting in a bodily fluid of said mammalautoantibodies to an epitope of the muscle specific tyrosine kinase,MuSK.

More specifically the neurotransmission disorder will preferably beMyasthenia gravis and more particularly a subclass or subtype of MGwhich is generally found in patients who do not exhibit the ability toimmunoprecipitate radiolabeled AChR with their bodily fluids.

This aspect of the invention is particularly advantageous because theidentification of this new subclass or subtype of MG patients will allowfor more accurate and speedy diagnosis of individuals by medicalpractitioners. The method according to this aspect of the invention willallow for detection of neurotransmission abnormalities that are eithercongenital or acquired, for example, postnatally or prenatally fromtransmission from the mother to the fetus. As set out in more detail inthe example provided, some mothers of babies with developmentaldisorders, such as paralysis and fixed joints were identified as havingantibodies to MuSK, which were transferred placentally.

Until now, MuSK has been studied primarily in NMJ development. Thepresence of antibodies to the extracellular domain of MuSK in anacquired disorder implies that MuSK is functional at the adult NMJ, andimplicates MuSK as a novel target for pathogenic autoantibodies causingMyasthenia gravis. The isolation and purification of this anti-MUSKautoantibody will give rise to a useful product which may be exploitableas an indicator of neurotransmission diseases.

Preferably, the method according to the first aspect of the invention,comprises the steps of a) contacting said bodily fluid with said MuSK oran antigenic determinant thereof; and b) detecting any antibody-antigencomplexes formed between said MuSK or an antigenic fragment thereof andantibodies present in said bodily fluid, wherein the presence of saidcomplexes is indicative of said mammal suffering from saidneurotransmission disorders.

The actual steps of detecting autoantibodies in a sample of bodilyfluids may be performed in accordance with immunological assaytechniques known per se in the art. Examples of suitable techniquesinclude ELISA, radioimmunoassays and the like. In general terms, suchassays use an antigen which may be immobilized on a solid support. Asample to be tested is brought into contact with the antigen and ifautoantibodies specific to the protein are present in a sample they willimmunologically react with the antigen to form autoantibody-antigencomplexes which may then be detected or quantitatively measured.Detection of autoantibody-antigen complexes is preferably carried outusing a secondary anti-human immunoglobulin antibody, typically anti-IgGor anti-human IgM, which recognizes general features common to all humanIgGs or IgMs, respectively. The secondary antibody is usually conjugatedto an enzyme such as, for example, horseradish peroxidase (HRP) so thatdetecting of autoantibody/antigen/secondary antibody complexes isachieved by addition of an enzyme substrate and subsequent calorimetric,chemiluminescent or fluorescent detection of the enzymatic reactionproducts.

Thus, in one embodiment the antibody/antigen complex may be detected bya further antibody, such as an anti-IgG antibody. Complexes mayalternatively be viewed by microscopy. Other labels or reportermolecules which may be used in a method according to the invention.Preferably, said reporter molecule or label includes any of a heavymetal, a fluorescent or luminescent molecule, radioactive or enzymatictag. Preferably, the label or reporter molecule is such that theintensity of the signal from the anti-human IgG antibody is indicativeof the relative amount of the anti-MuSK autoantibody in the bodily fluidwhen compared to a positive and negative control reading.

An alternative method of detecting autoantibodies for MuSK or an epitopethereof relies upon the binding of a MuSK or its epitope, together witha revealing label, to the autoantibodies in the serum or bodily fluid.This method comprises contacting MuSK or an epitope or antigenicdeterminant thereof having a suitable label thereon, with said bodilyfluid, immunoprecipitating any antibodies from said bodily fluid andmonitoring for said label on any of said antibodies, wherein thepresence of said label is indicative of said mammal suffering from saidneurotransmission or developmental disorder. Preferably, the label is aradioactive label which may be I, or the like. Iodination andimmunoprecipitation are standard techniques in the art, the details ofwhich may be found in references (4 and 6).

In a further aspect of the invention, there is provided an assay kit fordiagnosing neurotransmission disorders in mammals comprising an epitopeof muscle specific tyrosine kinase (MuSK) and means for contacting saidMuSK with a bodily fluid from a mammal. Thus advantageously, an assaysystem for detecting neurotransmission disorders, and particularlyMyasthenia gravis in patients who are anti-AChR autoantibody negative(AAAN) is provided. Prior to the present invention there was no basisfor providing an immediate clinical diagnosis for such patients.

Also provided by the invention is an isolated or purified autoantibodyspecific for MuSK. Such an antibody can be detected in bodily fluids ofmammals and isolated or purified therefrom using techniques which wouldbe known to the skilled practitioner, such as, immunoabsorption, orimmunoaffinity chromatography or high pressure chromatography.

In a further aspect the invention also comprises an isolated or purifiedantibody specific for an anti-MuSK autoantibody from bodily fluid of amammal. Such a purified or isolated antibody which is specific foranti-MuSK autoantibody may advantageously be used as a medicament, or inthe preparation of a medicament for treating neurotransmission disordersin a mammal, and preferably a human suffering from Myasthenia gravis.Such an antibody may also be included in a pharmaceutical compositiontogether with a pharmaceutically acceptable carrier, excipient ordiluent therefor. Antibodies, polyclonal or monoclonal may be preparedusing techniques which are known in the art. For example, the techniquedescribed by Kohler & Milstein (1975, Nature 256: 495-497) fordeveloping hybridomas capable of producing monoclonal antibodies may beused. Monoclonal antibodies for therapeutic use may be human monoclonalantibodies or chimeric human-mouse monoclonal antibodies. Chimericantibody molecules may be prepared containing a mouse antigen bindingdomain with human constant regions (Morrison et al., 1984, Proc. Natl.Acad. Sci. USA 81: 6581, Takeda et al., 1985, Nature 314: 452). Forproduction of antibody various host animals can be immunized byinjection with anti-MuSK autoantibody, or a fragment or derivativethereof, including but not limited to rabbits, mice, rats, etc. Variousadjuvants may be used to increase the immunological response, dependingon the host species, and including but not limited to Freund's (completeand incomplete), mineral gels such as aluminum hydroxide, surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanins, dinitrophenol, andpotentially useful human adjuvants such as BCG (Bacille Calmette-Guerin)and Corynebacterium parvum.

The present invention includes not only complete antibody molecules butfragments thereof. Antibody fragments which contain the idiotype of themolecule can be generated by known techniques, for example, suchfragments include but are not limited to the F (ab′)₂ fragment which canbe produced by pepsin digestion of the antibody molecule; the Fab′fragments which can be generated by reducing the disulfide bridges ofthe F (ab′)₂ fragments and the Fab fragments which can be generated bytreating the antibody molecule with papain and a reducing agent.

The antibody which is specific for anti-MuSK autoantibodies may also,advantageously, be used in a diagnostic kit for detectingneurotransmission disorders, such as Myasthenia gravis. Asaforementioned any protein which binds to the autoantibody may also beused such as an epitope or fragment of the MuSK protein itself. Such akit comprises an isolated or purified antibody specific for anti-MuSKautoantibody according to the invention and means for contacting saidantibody with a bodily fluid of a said mammal.

In accordance with the present invention a bodily fluid should be takento mean plasma, serum, whole blood, urine, sweat, lymph, faeces,cerebrospinal fluid or nipple aspirate. In general, however, the methodsof the invention will be performed on samples of serum or plasma.

In the pharmaceutical composition of the invention, preferredcompositions include pharmaceutically acceptable carriers including, forexample, non-toxic salts, sterile water or the like. A suitable buffermay also be present allowing the compositions to be lyophilized andstored in sterile conditions prior to reconstitution by the addition ofsterile water for subsequent administration. The carrier can alsocontain other pharmaceutically acceptable excipients for modifying otherconditions such as pH, osmolarity, viscosity, sterility, lipophilicity,solubility or the like. Pharmaceutical compositions which permitsustained or delayed release following administration may also be used.

The antibody or the MuSK protein or fragment thereof or thepharmaceutical composition of the invention may be administered orally.In this embodiment the antibody, MuSK or its eptopic fragment, orpharmaceutical composition of the invention may be encapsulated and/orcombined with suitable carriers in solid dosage forms which would bewell known to those of skill in the art.

Furthermore, as would be appreciated by the skilled practitioner, thespecific dosage regime may be calculated according to the body surfacearea of the patient or the volume of body space to be occupied,dependent on the particular route of administration to be used. Theamount of the composition actually administered will, however, bedetermined by a medical practitioner based on the circumstancespertaining to the disorder to be treated, such as the severity of thesymptoms, the age, weight and response of the individual.

In a further aspect, the present invention comprises a method oftreating a patient suffering from a neurotransmission disorder such asMyasthenia gravis comprising administering to said patient an effectiveamount of an antibody according to the invention or a MuSK protein or anepitope thereof.

In an even further aspect, the invention comprises a method for making apharmaceutical formulation for the treatment of neurotransmissiondisorders, comprising the steps of isolating or purifying an antibody orMuSK protein or fragment thereof according to the invention,manufacturing bulk quantities of said antibody and formulating theantibody in a compound including a pharmaceutically acceptable carrier,diluent or excipient therefor.

In an even further aspect, the invention comprises a method ofidentifying compounds capable of alleviating or treatingneurotransmission disorders, comprising the steps of contacting acandidate compound in the presence of MuSK or an epitope thereof and anantibody capable of binding MuSK, wherein a compound that preventsbinding of said antibody to MuSK or an epitope thereof is a candidatefor treating neurotransmission disorders. Such compounds may also beused in treating neurotransmission or developmental disorders or in themanufacture of a medicament for treating such disorders. The compoundsidentified may also, as would be appreciated by those of skill in theart, serve as lead compounds for the development of analogue compounds.The analogues should have a stabilized electronic configuration andmolecular conformation that allows key functional groups to be presentedto the polypeptides of the invention in-substantially the same way asthe lead compound. In particular, the analogue compounds have spatialelectronic properties which are comparable to the binding region, butcan be smaller molecules than the lead compound, frequently having amolecular weight below about 2 kD and preferably below about 1 kD.Identification of analogue compounds can be performed through use oftechniques such as self-consistent field (SCF) analysis, configurationinteraction (CI) analysis, and normal mode dynamics analysis. Computerprograms for implementing these techniques are available; e.g., Rein,Computer-Assisted Modeling of Receptor-Ligand Interactions (Alan Liss,New York, 1989). Methods for the preparation of chemical derivatives andanalogues are well known to those skilled in the art and are describedin, for example, Beilstein, Handbook of Organic Chemistry, Springeredition New York Inc., 175 Fifth Avenue, New York, N.Y. 10010 U.S.A. andOrganic Synthesis, Wiley, New York, USA. Furthermore, said derivativesand analogues can be tested for their effects according to methods knownin the art; see also supra. Furthermore, peptidomimetics and/or computeraided design of appropriate derivatives and analogues can be used.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention may be more clearly understood with reference tothe following examples and accompanying Figures wherein:

FIG. 1 is an illustration of the results obtained using antibodies fromAAAN patients reacting with the extracellular domain of MuSK. Samplesfrom AAAN patients are indicated as SNMG (sero-negative MG) as it waspreviously known.

a) The MuSK constructs used are shown in FIG. 1 a.

b) AAAN plasmas bound to COS-cells expressing full length MuSK(AAAN/MuSK). MuSK immunoreactivity appeared as a speckled pattern,similar to that seen previously with rabbit anti-MuSK antibodies (13).Non-transfected cells in the same field, demonstrated below by phasecontrast microscopy (arrows), showed non-specific binding only. Therewas no specific binding of AAAN plasmas to cells expressing MuSK lackingthe extracellular domains (MuSK D) or binding of healthy control plasma(HC/MuSK).

c) Two AAAN plasmas, but not a healthy control plasma,immunoprecipitated MuSK from detergent extracts of COS-cells expressingMuSK, and C2C12 myotubes. MuSK was identified by binding of anaffinity-purified rabbit anti-MuSK. It appears as a 110 kD band fromCOS-cells and as several bands representing different MuSK splicevariants in the C2C12 cells.

FIG. 2 is an illustration of results obtained by using IgG antibodies tothe extracellular domains of MuSK in seronegative MG measured by ELISA.a, Anti-MuSK antibodies were found in 17/24 AAAN patients compared with13 controls. Negative or borderline values only were found in 39anti-AChR positive MG patients. Non-specific binding of IgG to theplates has been subtracted. b, Titration of one AAAN plasma againstdifferent domains of MuSK. The antibodies bound strongly to MuSKconstructs expressing the distal immunoglobulin like domains, Igl-4 andIgl-2 (see FIG. 1 a), but not to the Ig3-4 membrane-proximal domains.

FIG. 3 is an illustration of the results that show that AAAN antibodiesinduce AChR clusters but inhibit agrin-induced AChR clustering.

a) In the absence of agrin, a moderate number of AChR clusters (asdemonstrated by rhodamine-a-bungarotoxin fluorescence) were inducedin-the presence of AAAN plasma compared to that in control plasma (HC).Agrin-induced clusters were found in the presence of healthy controlplasma but were inhibited in the presence of AAAN plasma.

b), c) The AChR clusters without (b) or with (c) added agrin in plasmaand IgG treated cultures. AAAN samples are labeled 1-6. Only theanti-MuSK positive plasmas and IgG preparations affected AChR clusters.

FIG. 4 is an illustration of the results obtained from further tests toconfirm the specificity of the test for Myasthenia gravis set out in theexamples provided.

FIG. 5 is an illustration of the results obtained from a test to detectMuSK antibodies in mothers of babies with development defects.

FIG. 6 is an illustration of the results obtained using an ELISA assayto detect MuSK antibodies in sera sent for analysis.

FIG. 7 is an illustration of the results obtained using animmunoprecipitation assay to detect MuSK antibodies in the sera of FIG.6.

FIG. 8 is correlation of the results of ELISA and immunoprecipitationassays of FIGS. 6 and 7 for detection of MuSK antibodies.

DETAILED DESCRIPTION OF THE INVENTION

A description of example embodiments of the invention follows.

EXAMPLE Patient Identification

Samples were obtained from 24 patients (18 F, 6 M) with moderate orsevere generalized MG, diagnosed by clinical electrophysiology, but inwhom the standard radioimmunoprecipitation assay for anti-AChRantibodies (4) was negative on several occasions. The age at onsetranged between 2 and 68 years (median 24) and the duration of symptomsat sampling was between one month and 13 years (median 1.0 year). In 18cases, plasma was obtained during therapeutic plasmapheresis whichimproved muscle strength. The remaining 6 samples were sera taken onfirst examination. Six of the patients had received corticosteroids forup to two months before sampling. Sera or plasmas were also obtainedfrom healthy volunteers and from patients with anti-AChR antibodypositive MG. IgG preparations were made using a Pierce ImmunoPureO (G)IgG purification kit.

MuSK and Agrin Expression Constructs.

Constructs encoding full length MuSK (13) and the soluble fragments-agrin ( 4/19) (20) have been described previously. MuSK deletionfragments comprising the entire extracellular domain (Igl-4; aa 1-490,numbers according to ref (10)) or the first half encomprising twoIg-domains (Igl-2; aa 1-230) were generated by insertion of artificialstop signals at these positions. N-terminal fragments of MuSK comprisingthe membrane-proximal extracellular domains, including Ig-domains 3 and4 (Ig3-4; aa 198-430), or the transmembrane region and intracellulardomain (MuSK D, aa 491-869) were generated. The correspondingc-DNA-fragments, including a newly introduced SphI-site, were linked toa vector containing an artificial signal sequence followed by sixhistidines and a 10aa epitope-tag (20). All constructs were transientlytransfected into COS7 cells (12). For the production of soluble agrinand MuSK constructs, cells were switched to serum-free medium the secondday after transfection. Conditioned media, containing MuSK or agrinfragments were removed 24 hours later and analyzed by Western blottingto confirm expression.

Immunostaining of MuSK-Transfected COS7 Cells.

COS7 cells were plated onto chamber slides the day after transfection.Two days later, cells were fixed with 2% paraformaldehyde and stained asdescribed (13). Plasmas of myasthenia gravis patients and controls wereanalyzed in various dilutions (between 1:20 and 1:5000). Boundantibodies were visualized with secondary antibodies conjugated to Cy3(anti-human IgG, Dianova). In all experiments, expression of transfectedMuSK constructs was confirmed by staining parallel slides withrabbit-anti MuSK antibodies (13).

Immunoprecipitation Experiments.

Detergent extracts were prepared from MuSK-transfected COS7 cells orfrom C2C12 myotubes that had been fused for five days. Theimmunoprecipitation was performed as described previously (12,13). AAANand control plasmas incubated with the extracts at 1:20. Rabbitanti-MuSK serum was used at 1:100. MuSK in the immunoprecipitates wasanalysed by Western blotting using affinity-purified serum antibodiesdirected against the a MuSK cytoplasmic sequence (13).

ELISA Detection of Anti-MuSK Antibodies.

Conditioned medium from MuSK-transfected'COS-cells or from control cellsmock-transfected with fish sperm DNA, was diluted 1:1 with 100 mMNaHC03-buffer, pH 9.5 and applied overnight to ELISA plates. Plasmaswere first tested at 1:5 in triplicates and subsequently at 1:10 induplicates. Bound antibodies were detected by horse radishperoxidase-protein A (Amersham) followed by o-phenylenediamine andmeasuring A492. For each sample, nonspecific immunoreactivity,determined by incubation of plates coated with conditioned medium frommock-transfected COS7 cells, was subtracted.

AChR Aggregation Assay.

The mouse muscle cell line, C2C12, was used to determine functionaleffects of antibodies. Cells were plated onto chamber slides, fused andtreated with or without agrin and/or plasmas or IgGs for five hours³.After fixation, AChRs were visualized with rhodamine-a-bungarotoxin andthe number of aggregates from more than 20 microscopic fields and atleast two independent cultures were measured as described (20).

Results

We initially looked for IgG antibodies in five AAAN plasmas and threeplasmas from healthy individuals using COS7 cells transfected with ratMuSK constructs (FIG. 1 a). The experiments were performed blind. Allfive AAAN plasmas (e.g., FIG. 1 b, AAAN), but none of the healthycontrol plasmas (e.g., HC), labeled MuSK aggregates on the cell surfaceat dilutions up to 1:1000. The pattern of immunoreactivity wasindistinguishable from labeling observed with antibodies raised againstrecombinant MuSK in rabbits. (13) Each of the AAAN plasmas recognizedthe extracellular domains of MuSK, since no immunoreactivity wasobserved with COS7 cells expressing the transmembrane and cytoplasmicdomains only (FIG. 1 b, MuSK D). Not all cells expressed MuSK (compareFIG. 1 b, AAAN/MuSK and Phase contrast, below), and thesenon-transfected cells and mock-transfected cells (not shown) did notbind the AAAN IgG antibodies.

Immunoprecipitation experiments confirmed that IgG antibodies in theAAAN plasmas recognized the native MuSK protein. Detergent extracts fromMuSK-expressing COS7 cells and from mouse C2C12 myotubes, that expressfunctional MuSK, were incubated with plasmas from two AAAN patients anda healthy control. Antibodies from both AAAN patients, but not from thecontrol, immunoprecipitated bands of 110 kDa that were identified asMuSK by binding of a specific anti-MuSK antibody (FIG. 1 c). With eachextract, similar-sized bands were immunoprecipitated by a rabbitanti-MuSK serum from parallel extracts (FIG. 1 c).

Sera and plasmas from AAAN, anti-AChR positive MG and healthyindividuals were then tested in an ELISA. Fragments comprising onlyextracellular domains of MuSK were expressed in COS7 cells from whichthese soluble constructs are secreted, and the media were used as asource of the polypeptide antigen. IgG anti-MuSK antibodies,substantially greater than the mean +3SDs of the healthy control values(0.08 OD units) were found in 17/24 AAAN samples, whereas onlyborderline or negative values were found in the anti-AChR positivepatients (FIG. 2 a). Four of the seven negative, compared with only twoof the 17 positive samples, were from patients who had receivedcorticosteroid therapy before sampling. Interestingly, in the 11patients tested in both assays, the OD values for binding of antibodiesto MuSK correlated (p<0.02) with IgG binding to the human TE671 cellline (which has features of human muscle) as measured previously (8).This suggests that MuSK is the target for AAAN IgG antibodies on theTE671 surface and that the negative values in seven samples are unlikelyto be due to a lack of reactivity with rat MuSK Further results withfour AAAN plasmas (e.g., FIG. 2 b) indicated that the majority ofantibodies are directed against the N-terminal sequences (constructIgl-2 in FIG. 1 a) and there was little reactivity with the membraneproximal half (construct Ig3-4 in FIG. 1 a). We found no evidence of IgMantibodies to MuSK (data not shown), suggesting that the target for theputative non-IgG antibodies reported previously in some of the AAANpatients (15) will still need to be defined.

To investigate functional effects of the MuSK autoantibodies, weexamined AChR clustering in myotubes derived from the mouse cell line,C2C12. In the absence of agrin (FIG. 3 a upper panels), the controlplasma produced very few clusters of AChRs (HC), whereas anti-MuSKpositive plasma induced AChR aggregates along the surface of themyotubes (AAAN). A similar antibody-induced induction of AChR-clusteringby artificial dimerization of the kinase has previously been reportedfor rabbit antibodies induced against purified MuSK (13). Strikingly,when agrin was added with the plasmas (FIG. 3 a, lower panels), themarked agrin-induced clustering which occurred in the presence ofcontrol plasma (HC) was not seen in the presence of AAAN plasmaindicating that the anti-MuSK antibodies had inhibited the agrin-inducedAChR clustering. Both the clustering (FIG. 3 b) and the inhibitoryactivity (FIG. 3 c) were found with each anti-MuSK positive plasmas orIgGs but not with anti-MuSK negative preparations. Since it is currentlyaccepted that agrin does not bind directly to MuSK, but via ahypothetical agrin-binding component called MASC (1,11), we speculatethat the antibodies in AAAN patients bind to MuSK in such a manner as toprevent its interaction with MASC. This interaction is known to dependon the N-terminal half of the extracellular domain of MuSK (16) which wefind to be the main target for the IgG antibodies in anti AChRautoantibody negative patients (FIG. 2 b).

To confirm the specificity of the test for myasthenia gravis, we testeda new group of controls (OND's) from patients with other neurologicaldisorders. (FIG. 4). Only one serum was borderline positive. Therelative incidence of MuSK antibodies in AAAN samples, was tested usinga second cohort (Cohort 2) of Myasthenia gravis patients who werenegative for acetylcholine receptor antibodies. All of these patientshad generalized disease and 11/16 of them were positive for MuSKantibodies.

Antibodies to the fetal isoform of the acetylcholine receptor are foundin a few mothers who have had babies born with complete paralysis andfixed joints (22,23). This severe condition is relatively common, butmaternal antibodies to fetal acetylcholine receptor are found in onlyabout 1% (Vincent, Dalton, unpublished findings). We asked whether MuSKantibodies might be present in some of these mothers. FIG. 5 shows, incomparison with the previously described results, that six mothers ofaffected babies out of a total of 200 tested (only 60 shown here) havethese antibodies in their serum. This indicates that each of these sixmothers has made an autoimmune response to MuSK and suggests that, aftertransfer of these antibodies across the placenta, they might be involvedin causing the babies' condition. Testing for antibodies to MuSK inmothers of babies with muscle paralysis and/or fixed joints mightindicate a fetal condition due to maternal antibodies.

To assess how the assay works out in practice, we have begun to compareresults from patients with definite SNMG or a strong suspicion of SNMGwith those in whom the diagnosis is questionable (? SNMG). FIG. 6 showsthat among the first group, which includes cohort 1 and cohort 2, theassay is positive in 39/66 and among those with a questionable diagnosisthe proportion is 6/25. The assay continues to be negative in healthyindividuals.

The ELISA assay used as identified in the above example is difficult tostandardize and we have tested an alternative assay, usingimmunoprecipitation of 5I-MuSK. For this test, the purifiedextracellular domain of MuSK is iodinated using 125I (carrier free fromAmersham as for bungarotoxin in Ref (4,6) or with chloramine T (standardconditions)). The iodinated MuSK is then separated from free 125I by gelfiltration. The 125I-MUSK (approximately 50,000 cpm) is then added to 10microlitres of the patient's serum over night. To immunoprecipitate thepatients' antibodies and any 125I-MuSK that is bound by them, excess ofa sheep antibody to human IgG is added. The precipitate is centrifugedto form a pellet, washed and counted for radioactivity. The results(FIG. 7) show that healthy controls precipitated less than 1200 cpm,whereas 38/66 of the SNMG patients precipitated over 1200 cpm, the valuerising to 7500 cpm which corresponds to approximately 1 nmole of MuSKprecipitated per liter of serum. The assay was also positive in 5/25patients with? SNMG.

The results of the ELISA and immunoprecipitation assays were highlycorrelated (FIG. 8). Most of the sera were positive with both assays ornegative with both assays; there were three sera that gave negativeresults with the immunoprecipitation and positive with ELISA, and twosera that were negative with the ELISA and positive with theimmunoprecipitation.

REFERENCES

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1. A method for diagnosing congenital and acquired muscle disordersrelated to muscle specific tyrosine kinase (MuSK) in a mammal comprisingthe step of detecting in a bodily fluid of said mammal autoantibodies toan epitope of muscle specific tyrosine kinase (MuSK).
 2. A methodaccording to claim 1 wherein said method comprises the steps of: a)contacting said bodily fluid with muscle specific tyrosine kinase (MuSK)or an antigenic determinant thereof; and b) detecting anyantibody-antigen complexes formed between said receptor tyrosine kinaseor an antigenic fragment thereof and antibodies present in said bodilyfluid, wherein the presence of said complexes is indicative of saidmammal suffering from said neurotransmission or developmental disorders.3. A method according to claim 2 wherein said antibody-antigen complexis detected using an anti-IgG antibody tagged or labeled with a reportermolecule.
 4. A method according to claim 3 wherein said reportermolecule or label includes any of a heavy metal, a fluorescent orluminescent molecule, radioactive or enzymatic tag.
 5. A methodaccording to claim 4 wherein said enzymatic tag comprises horseradishperoxidase-protein A followed by reaction with o-phenylenediamine forsubsequent measurement at A492.
 6. A method according to claim 3 wherebythe intensity of the signal from the anti-human IgG antibody isindicative of the relative amount of the anti-MuSK autoantibody in thebodily fluid when compared to a positive and negative control reading.7. A method according to claim 1, comprising contacting MuSK or anepitope or antigenic determinant thereof having a suitable labelthereon, with said bodily fluid, immunoprecipitating any antibody/MuSKcomplex or antibody/MuSK epitope or antigenic determinant complex fromsaid bodily fluid and monitoring for said label on any of saidantibody/MuSK complex or antibody/MuSK epitope or antigen determinantcomplex, wherein the presence of said label is indicative of said mammalis suffering from said neurotransmission or developmental disorderrelated to muscle specific tyrosine kinase (MuSK).
 8. A method accordingto claim 7 wherein said label is a radioactive label.
 9. A methodaccording to claim 8 wherein said label is ¹²⁵I.
 10. A method fordiagnosing congenital and acquired muscle disorders related tointerference of the agrin/MuSK/AChR pathway within a mammal comprisingthe step of detecting in a bodily fluid of said mammal autoantibodies toan epitope of muscle specific tyrosine kinase (MuSK).
 11. A method ofdiagnosing myasthenia gravis related to muscle specific tyrosine kinase(MuSK) in a mammal comprising the step of detecting autoantibodies to anepitope of muscle specific tyrosine kinase (MuSK) in a bodily fluid ofsaid mammal.
 12. The method of claim 11, wherein the bodily fluid isselected from the group consisting of plasma, serum, whole blood, urine,sweat, lymph, faeces, cerebrospinal fluid or nipple aspirate.